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目的建立针对以血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR-2)为靶标的特异及灵敏的细胞模型体系,用于酪氨酸激酶受体(receptor tyrosine kinase,RTK)抑制剂的筛选和研究。方法构建人源VEGFR-2真核表达载体pcDNA3.1-VEGFR-2,将其转染至NIH 3T3细胞,获得稳定转染的细胞克隆,并对其进行功能学鉴定及应用;应用瞬时转染VEGFR-2的HeLa细胞,建立VEGF依赖性的受体磷酸化细胞模型及应用。结果在无血清培养条件下,稳定转染VEGFR-2的NIH 3T3细胞获得VEGF依赖性的细胞增殖特征;HeLa细胞经瞬时转染VEGFR-2后,可以检测出明显的VEGF依赖性的受体酪氨酸磷酸化。通过应用已知VEGFR-2抑制剂及自行合成化合物,确证以上细胞模型具有较高机制针对性的特征。结论所构建并确证的细胞模型具有较高特异性和灵敏性,可适用于VEGFR-2和其他RTK抑制化合物的体外筛选与研究。
Objective To establish a specific and sensitive cell model targeting to vascular endothelial growth factor receptor-2 (VEGFR-2) as target for tyrosine kinase receptor (RTK ) Inhibitors screening and research. Methods The human VEGFR-2 eukaryotic expression vector pcDNA3.1-VEGFR-2 was constructed and transfected into NIH 3T3 cells. The stable transfected cell clone was obtained and its function was identified and applied. Transient transfection VEGFR-2 HeLa Cells, Establishing a VEGF-dependent Receptor Phosphorylation Cell Model and Its Application. Results Under the condition of serum-free culture, VEGFR-2-stably transfected NIH 3T3 cells obtained the characteristics of VEGF-dependent cell proliferation. After transient transfection of HeLa cells with VEGFR-2, obvious VEGF- Amino acid phosphorylation. The above cell model is confirmed to have a high degree of mechanism-specific characteristics by the use of known VEGFR-2 inhibitors and self-compound synthesis. CONCLUSION: The constructed and confirmed cell model is highly specific and sensitive and suitable for the in vitro screening and research of VEGFR-2 and other RTK inhibitory compounds.